Serum granulosa cell-derived TNF-α promotes inflammation and apoptosis of renal tubular cells and PCOS-related kidney injury through NF-κB signaling.

Polycystic ovary syndrome (PCOS) is a disorder with endocrinal and metabolic problems in reproductive aged women. Evidence shows that PCOS is in a high prone trend to develop kidney diseases. In this study, we investigated the mediators responsible for PCOS-related kidney injury. We found that tumor necrosis factor (TNF-α) levels were significantly increased in serum and primary cultured granulosa cells (GCs) from PCOS patients. Serum TNF-α levels were positively correlated with serum testosterone and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, suggesting its positive role in the severity of PCOS. Serum TNF-α levels were also positively correlated with the levels of urinary KapU, LamU, α1-MU and ß2-MU, the markers for renal tubular cell-derived proteinuria. We established a PCOS mouse model by resection of the right kidney, followed by daily administration of dihydrotestosterone (DHT, 27.5 µg, i.p.) from D7 for 90 days. We found that TNF-α levels were significantly increased in the ovary and serum of the mice, accompanied by increased renal tubular cell apoptosis, inflammation and fibrosis in kidneys. Furthermore, the receptor of TNF-α, tumor necrosis factor receptor 1 (TNFR1), was significantly upregulated in renal tubular cells. We treated human ovarian granulosa-like tumor cells (KGN) with DHT (1 µg/ml) in vitro, the conditioned medium derived from the granulosa cell culture greatly accelerated apoptotic injury in human proximal tubular epithelial cells (HKC-8), which was blocked after knockdown of TNF-α in KGN cells. Furthermore, knockdown of TNFR1 in renal tubular epithelial cells greatly ameliorated cell injury induced by granulosa cell-derived conditioned medium. These results suggest that serum TNF-α plays a key role in mediating inflammation and apoptosis in renal tubular cells associated with PCOS-related kidney injury.

Enantioselective Synthesis of Fused Butyrolactones via Organocatalytic Formal [2 + 2 + 2] Annulation of γ-Butenolides, Methylene Indolinones, and Nitroolefins.

An organocatalyzed asymmetric synthesis of fused butyrolactones via formal [2 + 2 + 2] annulation between γ-butenolides, methylene indolinones, and nitroolefins in a one-pot process has been established. Products containing six contiguous stereocenters could be obtained in good yields (up to 95%) with excellent enantioselectivities (up to >99% ee) catalyzed by a l-tert-leucine-derived bifunctional thiourea catalyst.

[Adsorption of Fulvic Acid on Virgin and Aging Microplastics].

Due to the wide distribution and strong adsorption ability of microplastics (MPs) for organic matter in aquatic environments, the interaction between MPs and natural organic matter (NOM) cannot be ignored. In this study, virgin and aging polyamide 66 (PA66) and polypropylene (PP) MPs were used to adsorb fulvic acid (FA) in order to understand the effect of MPs on NOM. The results indicated that the kinetics experimental data of FA adsorption on virgin and aging MPs well fitted the pseudo-second-order model (R2>0.94), and the adsorption equilibrium was reached at 48 h. Compared to that of PP, the adsorption capacity of FA on PA66 were relatively higher, and the aging process improved the adsorption ability of MPs for FA. Freundlich models were well fitting with the adsorption isotherms experimental data compared to Langmuir models, indicating that the adsorption of FA on the virgin and aging MPs was a multi-layer heterogeneous physical process. The thermodynamics analysis revealed that the adsorption was spontaneous and endothermic. With the increase in pH, the adsorption capacity of FA first decreased and then increased. The desorption experiment indicated that the FA desorbed from the tested MPs in ultrapure water obtained higher desorption rates than that in surface water, and the desorption rates of aging MPs were less than that of the corresponding virgin ones. The aging process had a great influence on the structure of MPs, which resulted in a distinct increase in surface area and roughness of MPs, but slightly affected functional groups. Specific surface area and polarity of MPs were the main influencing factors for the adsorption process, and the main mechanism of FA adsorption on the tested MPs was hydrophobic and π-π interactions.

Nanostructure-based surface-enhanced Raman scattering for diagnosis of cancer.

Cancer is a malignant disease that seriously affects human health and life. Early diagnosis and timely treatment can significantly improve the survival rate of cancer patients. Surface-enhanced Raman scattering (SERS) is an optical technology that can detect and image samples at the single-molecule level. It has the advantages of rapidity, high specificity, high sensitivity and no damage to the sample. The performance of SERS is highly dependent on the properties, size and morphology of the SERS substrate. Preparation of SERS substrates with good reproducibility and chemical stability is a key factor in realizing the wide application of SERS technology in cancer diagnosis. In this review we provide a detailed presentation of the latest research on SERS in cancer diagnosis and the detection of cancer biomarkers, mainly focusing on nanotechnological approaches in cancer diagnosis by using SERS. We also consider the future development of nanostructure-based SERS in cancer diagnosis.

[Response of nutrient release and ecological stoichiometry of litter to simulated nitrogen deposition in evergreen broad-leaved forest in central Yunnan, China]. / æ»‡ä¸­å¸¸ç»¿é˜”å¶æž—å‡‹è½ç‰©å »åˆ†é‡Šæ”¾åŠç”Ÿæ€åŒ–å­¦è®¡é‡ç‰¹å¾å¯¹æ¨¡æ‹ŸN沉降的响应.

We examined nutrient release and ecological stoichiometric characteristics of litters under N deposition in an evergreen broadleaved forest in Mopan Mountain in central Yunnan. Nylon net bag method was used for in situ decomposition of leaf litter and twig litter. There were four treatments, including control (CK, 0 g N·m-2·a-1), low nitrogen (LN, 5 g N·m-2·a-1), medium nitrogen (MN, 15 g N·m-2·a-1), and high nitrogen (HN, 30 g N·m-2·a-1). The results showed that after one year of N addition, the contents of C and N in leaf litter, twig litter and soil increased gradually with the increases of N addition rates, with increases of 0.3%-8.2% and 4.9%-69.0%, respectively. C/N gradually decreased with increasing N addition rates, with a decrease of 0.8%-37.8%. There was no significant difference in P content, C/P and N/P of twig litter under different treatments. Treatment duration and N application rate significantly affected the N and P contents and stoichiometric ratios of leaf litter, twig litter and soil. During the 1-year decomposition process, the residual rates of C, N and P in litters were successively in the modes of release, leaching-enriched-released and leaching-enriched. Exogenous N addition significantly inhibited the release process of C, N and P in litter. The contents of C and P in soil were significantly positively correlated with the contents of N and P in litter, while the contents of N in soil were significantly positively correlated with the contents of C and N in litter. There was a significant correlation of stoichiometric characteristics between litter and soils of evergreen broadleaved forest under N deposition. Our results were helpful to understand the response mechanism of litter decomposition process of forest ecosystem to N deposition.

Fusapyrone A, a γ-pyrone derived from a desert Fusarium sp.

In this work, we isolated and characterized fusapyrone A (1), a new γ-pyrone derivative, along with six previously described compounds from the rice fermentation of Fusarium sp. CPCC 401218, a fungus collected from the desert. The structure of 1 was characterized using various spectroscopic analyses, such as MS, IR, 1D, and 2D NMR. The absolute configuration of 1 was determined through the use of 13C NMR chemical shifts, electronic circular dichroism (ECD) and optical rotation (OR) calculations. Compound 1 was found to have weak antiproliferative activity for Hela cells, with an IC50 of 50.6 µM.[Formula: see text].

Applications of surface-enhanced Raman spectroscopy in detection fields.

Surface-enhanced Raman spectroscopy (SERS) is a Raman spectroscopy technique that has been widely used in food safety, environmental monitoring, medical diagnosis and treatment and drug monitoring because of its high selectivity, sensitivity, rapidness, simplicity and specificity in identifying molecular structures. This review introduces the detection mechanism of SERS and summarizes the most recent progress concerning the use of SERS for the detection and characterization of molecules, providing references for the later research of SERS in detection fields.

CD8dimCD3+ lymphocytes in fever patients might be biomarkers of active EBV infection and exclusion indicator of T-LGLL.

Background: Massive monoclonal or oligoclonal expansion of CD8+ T cells is a notable feature of primary infections of the Epstein-Barr virus (EBV). However, the clinical significance of this expansion is not clear. Results: An increase in the CD8dimCD3+ lymphocyte subset in patients with active EBV infection was due to caspase-8-dependent apoptosis was found using flow cytometry in this study. The number of these cells was associated with the illness severity. Pan-T-cell antigen and receptor analyses were also compared in patients with active EBV infections and T-cell large granular lymphocytic leukemia to provide additional diagnostic information. Conclusion: The increase in CD8dimCD3+ cells could be a biomarker of active EBV infection and an exclusion indicator of T-cell large granular lymphocytic leukemia with flow cytometric analysis.

[Sorption Behaviors of Copper Ions and Tetracycline on Microplastics in Aqueous Solution].

The interaction between microplastics, heavy metals, and antibiotics can lead to combined pollution, which could result in greater environmental damage. The pathway and mechanism of the interaction between microplastics, heavy metals, and antibiotics are the preconditions for evaluating the associated environmental risk; however, these are not well understood. As probe sorbates, the sorption behaviors of copper ions (Cu2+) and tetracycline (TC) on two microplastics [high density polyethylene (HPDE) and general-purpose polystyrene (GPPS)] in aqueous solution were investigated and the welding theory with relevant experimental results were discussed. The adsorption capacity of HDPE was greater than that of GPPS in a single Cu solution, whereas the reverse situation occurred in a single TC solution. Moreover, the adsorption capacity of the microplastics in a Cu2+-TC binary solution was larger than that in the single solutions. The pseudo-second-order kinetic models to describe the adsorption process were reasonable and the entire process could be divided into two phases:surface adsorption and internal diffusion. The Langmuir model provided a better fit of the data than did the Freundlich model. In the single solutions, the saturated adsorption amounts of Cu2+ and TC were 0.178 µmol·g-1 and 0.257 µmol·g-1, respectively, for GPPS, and 0.334 µmol·g-1 and 0.194 µmol·g-1, respectively, for HDPE. In the binary solution, the corresponding numerical values were 0.529 µmol·g-1 and 0.411 µmol·g-1, respectively, for GPPS and 0.471 µmol·g-1 and 0.341 µmol·g-1, respectively, for HDPE. The variations in the surface morphological characteristics and chemical functional groups were the main reasons for the difference in the adsorption behavior of microplastics. The variation of the pH of the adsorption system could change the existing forms and surface electrical properties of microplastics and adsorbed objects, and subsequently affected the equilibrium adsorption capacity. When the ambient temperature was in the range of 15 to 35℃, increasing the temperature was unfavorable for the adsorption process. Cu2+ and TC could produce a synergistic effect under the conditions of coexistence. The formation of complexes and bridging make Cu2+ and TC more easily adsorbed by microplastics.

[Occurrence and Prediction Model of Disinfection By-Products in Tap Water].

Disinfection by-products (DBPs) are defined as important parameters that can deteriorate drinking water quality. The investigation was performed at a laboratory located on a campus in H City of the Zhejiang province. The purpose of the work was to obtain knowledge on the occurrence of DBPs in tap water and boiled water taken from the same pipe, to establish a statistical model to predict DBPs information in tap water based on physicochemical parameters, and to evaluate carcinogenic and non-carcinogenic risks caused by DBPs on a predictional level. The results showed three categories of trihalomethanes (THMs), haloacetonnitrile (HANs), and haloacetic acids (HAAs), including 10 species of disinfection by-products detected in drinking water. The detection rate of target DBPs in tap water was 100% and the concentrations varied in the ranges of 10.12-28.39, 0.98-5.19, and 2.65-7.83 µg·L-1, respectively. In boiled water, bromochloracetonitrile (BCAN) was not detected; the detection rates of tribromomethane(TBM), trichloroacetonitrile (TCAN), and dibromoacetonitrile (DBAN) were 46.43%, 82.14%, and 92.86%, respectively, while the detection rate for other DBPs was 100%. The concentrations of THMs, HANs, and HAAs were in the ranges of 0.60-12.58, 0.02-0.52, and 2.42-5.86 µg·L-1, respectively. After heating, the concentrations of THMs and HANs decreased by 84.22% and 91.45%, respectively. No obvious decrease was found for HAAs. The pH value and specific ultraviolet absorbance (SUVA) had positive correlation with DBPs, whereas residual chlorine and ammonia nitrogen had negative correlation with DBPs. Based on the correlation between the physicochemical parameters and DBPs, a multiple linear regression prediction model of THMs was established, with deviation less than 10.00%, which can be used for the prediction of THMs in tap water. Based on the EPA recommended health risk assessment model, the carcinogenic and non-carcinogenic risks of chlorine disinfection by-products through oral intake were calculated. It was found that the carcinogenic risks caused by the disinfection by-products in the tap and boiled water were (17.24-84.63)×10-6 and (25.49-258.82)×10-7, respectively, and the non-carcinogenic risks were (4.17-50.32)×10-2 and (6.52-107.74)×10-3, respectively. The carcinogenic risk caused mainly by THMs and bromodicloromethane (BDCM) contributed the highest cancer risk in tap water, while for boiled water, trichloromethane (TCM) was found to contribute the highest cancer and non-carcinogenic risk. In boiled water, the reduction of THMs was up to 94.38%, and the cancer risk was reduced by 79.00%.

[Responses of litter decomposition in two subalpine plantations to simulated nitrogen deposition in central Yunnan, China]. / 滇中亚高山人工林凋落物分解对模拟氮沉降的响应.

From February 2018 to January 2019, a field experiment of simulated nitrogen (N) depo-sition was conducted in Pinus armandii and Pinus yunnanensis plantations in the subalpine region of central Yunnan, China. The litterbag method was used for in situ litter (leaf and twig) decomposition experiment in both plantations. Four levels of N addition were applied, i.e., control (CK, 0 g N·m-2·a-1), low nitrogen (LN, 5 g N·m-2·a-1), medium nitrogen (MN, 15 g N·m-2·a-1), and high nitrogen (HN, 30 g N·m-2·a-1). The results showed that the annual decomposition rates of leaf and twig in P. armandii were 34.8% and 18.0%, which were higher than the 32.2% (leaf) and 16.1% (twig) in P. yunnanensis. Under N deposition, the LN treatment reduced the time of 95% mass loss of leaf and twig litter in P. armandii by 0.202 and 1.624 years, the MN treatment reduced by 0.045 and 1.437 years, and the HN treatment increased by 0.840 and 2.112 years, respectively. In the P. yunnanensis plantation, the LN treatment reduced the time of 95% mass loss of leaf and twig litter by 0.766 and 4.053 years, while the MN treatment increased by 0.366 and 0.455 years, and the HN treatment increased by 0.826 and 0.906 years, respectively. Litter (leaf and twig) decomposition of both P. armandii and P. yunnanensis were promoted by low N treatment and inhibited by high N treatment. The effects of N deposition on litter decomposition of two plantations were significantly correlated with the contents of cellulose and lignin in litter. In conclusion, the responses of litter decomposition to N deposition mainly depended on the litter substrate, especially cellulose and lignin contents.

Mesenchymal Stem Cell-Platelet Aggregates Increased in the Peripheral Blood of Patients with Acute Myocardial Infarction and Might Depend on the Stromal Cell-Derived Factor 1/CXCR4 Axis.

Bone marrow mesenchymal stem cells (MSCs) are a rare subset of nonhematopoietic progenitor cells and are appealing biomaterial for multiple tissue damage repairs. Transplantation of MSCs is proved to improve heart function after myocardial ischemia. However, the limitations of MSC injection approaches are equally obvious. As a multiple-function cell, platelets (PLTs) are also known playing important roles in cardiac recovery after myocardial infarction. In this study, we analyzed circulating MSC-PLT aggregate numbers in acute myocardial infarction (AMI) patients by flow cytometry. We found more MSC-PLT aggregates in patients with AMI than in healthy controls, and the patients with higher MSC-PLT aggregates had better prognosis. When stromal cell-derived factor 1 (SDF-1) binds to its receptor CXC chemokine receptor 4 (CXCR4), they play an important role in MSC migration and engraftment. We explored SDF-1 and CXCR4 expression on PLT surface by flow cytometry and found relative mean fluorescence intensity of PLT CXCR4 and the number of MSC-PLT aggregates showed a significant correlation. Meanwhile, in vitro experiments demonstrated that SDF-1/CXCR4 was crucial in MSC-PLT aggregate formation, which might suggest a novel mechanism that SDF-1/CXCR4 is involved in MSCs homing and myocardial repair after AMI. There may be another strategy to encourage myocardial repair in AMI patients by increasing the expression of SDF-1 on MSCs and promoting the formation of MSC-PLT aggregates.

[Lactic acid inhibits the formation of semen-derived amyloid fibrils].

OBJECTIVE: To investigate the inhibitory effect of lactic acid on semen-derived amyloid (SEVI) fibril formation. METHODS: PAP248-286 (2 mg/mL) was incubated with 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 mg/mL of lactic acid. After incubation for different times, aliquots were drawn from each sample for Thioflavin T (ThT) and Congo red staining to monitor semen-derived amyloid fibril formation. The ß sheet structure formation of PAP248-286 was measured by circular dichroism spectrum, and the morphology of amyloid fibrils incubated with or without lactic acid was observed with transmission electron microscopy (TEM). The enhancing effect of amyloid fibril incubated with lactic acid at different time points was determined using virus infection assay. PAP248-286 (2 mg/mL) was incubated with dilutions of vaginal secretion from healthy women, and amyloid fibril formation was detected with ThT and Congo red staining. RESULTS: Lactic acid inhibited SEVI fibril formation in a dose-dependent manner in vitro. Lactic acid at 0.5 mg/mL completely inhibited 2 mg/mL SEVI fibril formation within 48 h. After incubation for 48 h, lactic acid at 1 mg/mL inhibited the formation of ß-sheet structure of SEVI (2 mg/mL) and completely inhibited 2 mg/mL PAP248-286 aggregation as observed with TEM. In the presence of lactic acid, PAP248-286 lost the ability to enhance virus infection. Vaginal secretion inhibited SEVI fibril formation in a dose-dependent manner, and virtually no SEVI fibril occurred after incubation of 2 mg/mL PAP248-286 with 67% vaginal secretion. CONCLUSION: Lactic acid inhibits SEVI fibril formation in vitro.

[Synthesis and antifungal activities of N-1,3,4-thiadiazol-2-yl-4-oxo-thiochroman-2-yl-formamide derivatives].

Thiochromanones and 1,3,4-thiadiazoles as heterocyclic compounds have broad biological activities. In order to find novel compounds with antifungal bioactivity, substituted thiophenol and maleic anhydride were used to synthesize the intermediate 4-oxothiochromane-2-carboxylic acid. It was reacted with 2-amino-1,3,4-thiadiazole to get fourteen target compounds containing 1,3,4-thiadiazole moiety. The structures of the obtained compounds were confirmed by 1H NMR, 13C NMR and HR-MS. All compounds were investigated for antifungal activity via microdilution broth method. The results showed that the target compounds 3a and 3c to Epidermophyton floccosum and Mucor racemosus exhibited better antifungal activity than the positive control fluconazole, in which the minimum inhibition concentration can reach 8 µg·mL−1 and 16 µg·mL−1. Compound 3e showed significant inhibitory activity to Helminthosporium maydis, Sclerotinia sclerotiorum and Botrytis cinerea compared with that of the positive control carbendazim. Compound 3b exhibited inhibitory activity to Helminthosporium maydis better than the positive control carbendazim.

Synthesis, Characterization and Antifungal Evaluation of Novel Thiochromanone Derivatives Containing Indole Skeleton.

Invasive fungal disease constitutes a growing health problem and development of novel antifungal drugs with high potency and selectivity against new fungal molecular targets are urgently needed. In order to develop potent antifungal agents, a novel series of 6-alkyl-indolo[3,2-c]-2H-thiochroman derivatives were synthesized. Microdilution broth method was used to investigate antifungal activity of these compounds. Most of them showed good antifungal activity in vitro. Compound 4o showed the best antifungal activity, which (inhibition of Candida albicans and Cryptococcus neoformans) can be achieved at the concentration of 4 µg/mL. Compounds 4b (inhibition of Cryptococcus neoformans), 4j (inhibition of Cryptococcus neoformans), 4d (inhibition of Candida albicans) and 4h (inhibition of Candida albicans) also showed the best antifungal activity at the concentrations of 4 µg/mL. The molecular interactions between 4o and the N-myristoyltransferase of Candida albicans (PDB ID: 1IYL) were finally investigated through molecular docking. The results indicated that these thiochromanone derivatives containing indole skeleton could serve as promising leads for further optimization as novel antifungal agents.

[Synthesis, biological activity and molecular docking research of N-{[(4-oxo-thiochroman-3-yl)phenyl]-methyl}acetamide derivatives as α-glucosidase inhibitors].

In order to develop potent antidiabetic agents that have inhibitory effect to a-glucosidase, twelve ß-acetamido ketone derivatives such as N-{[(substituted-4-oxo-thiochroman-3-yl)phenyl]-methyl}acetamide are designed and synthesized through one-pot Dakin-West reaction. Their chemical structures are confirmed by 1H NMR, 13C NMR, IR and HR-MS. In vitro α-glucosidase inhibition assays of compounds 4a-41 were carried out using glucose oxidase method. The result indicated that most of them possess inhibitory activity in vitro. Compound 4k showed the most potent inhibitory activity with 87.3% inhibition of α-glucosidase at the concentration of 5.39 mmol x L(-1). The structure-activity relationship of these ß-acetamido ketone derivatives was discussed preliminarily. Moreover, the molecular docking method was used to study the interaction mode of compound 4k and α-glucosidase. Our results will be helpful for designing of α-glucosidase inhibitors in the future.

One-pot three-component synthesis of 3-hydroxy-5,5-dimethyl-2-[phenyl(phenylthio)methyl]cyclohex-2-enone derivatives under ultrasound.

3-Hydroxy-5,5-dimethyl-2-[phenyl(phenylthio)methyl]cyclohex-2-enone is synthesized via one-pot three-component reactions of aromatic aldehyde, substituted thiophenol and 5,5-dimethyl-1,3-cyclohexanedione catalyzed by p-dodecylbenzene sulfonic acid (DBSA) under ultrasound. Under ultrasound irradiation the yields are much higher (sometimes substantially, by almost double) and the reaction time decreases substantially, the reaction conditions are milder. This method provides several advantages such as environment friendliness, high yields and simple work-up procedure and the protocol provides a novel alternative for the synthesis of thioether.

Ionic liquid catalyzed synthesis of 2-(indole-3-yl)-thiochroman-4-ones and their novel antifungal activities.

2-(Indole-3-yl)-thiochroman-4-ones were synthesized via ionic liquid and tested for in vitro antifungal activity. The contribution of ionic liquid to Michael addition reaction is significant. Structures of all compounds are elucidated by (1)H NMR, (13)C NMR and HRMS. Most of these compounds showed better antifungal activity than fluconazole. The results suggest that 2-(indole-3-yl)-thiochroman-4-ones would be efficient antifungal agents.

Synthesis and pharmacological evaluation of novel bisindolylalkanes analogues.

In an effort to develop potent anti-cancer chemopreventive agents that act on topoisomerase II, a novel series of bisindolylalkanes analogues such as 3,3'-(thiochroman-4,4-diyl)bis(1H-indole) are synthesized. Structures of all compounds are elucidated by (1)H NMR, (13)C NMR and HRMS. Anti-proliferative activities for all of these compounds are investigated by the method of MTT assay on 7 human cancer lines. Most of them showed antitumor activities in vitro, the half maximal inhibitory concentration (IC50) value is 7.798 µg/mL of 3a against MCF7. Compound 3a showed comparable topoisomerase II inhibitory activity to etoposide (VP-16) at 100 µM concentration. The rest of the compounds also showed varying degree topoisomerase II inhibitory activity.

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis.

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.